Topic P02: Diagnostics of streptococci To study: Streptococcus (from textbooks, www etc.) From spring term: Microscopy, culture, biochemical identification, neutralization Table for major results of Task 1 to Task 5 (to be filled step by step): Strain K L M N P Q R S Gram stain – Task 1 Catalase test Task 2a Slanetz-Bartley medium – Task 2b Culture (blood agar) Task 3 Size Colour Shape Profile Agar changes Other PARTIAL CONCLUSION Task 4a: Optochin (viridans strep only) 4b: STREPTOtest 16 (oral strep only) Task 5a: PYR test (haem. strep only) Task 5b: CAMP (haem. strep only) Task 5c: Agglutina- tion (nAnB only) FINAL CONCLUSION Task 1: Microscopy of suspicious strains There are letter-labelled strains on the table. Gram-stain them and assess which one is NOT a Gram-positive coccus. To avoid confusion, label the slides using a marker. Write your results in the table. Task 2: Basic culture and biochemical tests – genus determination a) Catalase test for the differentiation from staphylococci Perform the catalase test with all the strains from Task 1 with the exception of the strain proven not to be a G+ coccus. Staphylococci should be catalase positive, streptococci and enterococci should be catalase negative. b) Growth on Slanetz-Bartley (SB) agar for the differentiation of enterococci The plate with SB agar has been inoculated with all the strains, each in one sector. However, only one of them is growing and that would be an enterococcus, not a streptococcus. Write the results of 2a and 2b in the table. Note: The same thing can be done with bile-aesculin medium, too, but the colour of colonies is different. Task 3: Blood agar culture The plates with blood agar again contain all strains. Observe all of them, but describe only the strains that were not excluded by tasks 1 and 2. Describe the colony morphology, and especially the haemolysis, partial haemolysis or viridation. Write your findings in the table. Now write “Partial conclusion” to your table. Write “NO STREP” (no streptococcus), “HAEM STREP” (partial or total haemolysis) or “VIR STREP” (viridation) to each strain K to S,. Task 4: More detailed diagnostics of streptococci with viridation a) Optochin test Your task is to evaluate the result of the optochin test in the two strains shown to be streptococci with viridation. The optochin test does not differ from a common diffusion disc test but the effective drug (optochin) is not used for treatment any longer. The strain with the presence of the inhibition zone around the optochin disc is S. pneumoniae, the strain without the zone is an “oral streptococcus”. Draw your result, and write “+” or “–” to the table. + = any susceptibility zone (not necessary to measure) – = no zone b) Biochemical determination of “oral” streptococcus In the strain found in Task 4a to be an “oral streptococcus”, evaluate the results of a biochemical microtest (STREPTOtest 16), using methods learned in the summer term. Tube First row with 8 wells Second row with 8 wells VPT 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification Streptococcus ___________ % of probability T index Task 5: Diagnostics of streptococci with partial or total haemolysis This task will be done with the three strains proven to be streptococci with haemolysis (parts a, b); the last part (c) will be only performed with the one proven to be “non-A-non-B” streptococcus. a) PYR test PYR test is a strip-test, similar to the oxidase test. For reading the colour result, it is necessary to wait for about five minutes, then add a drop of “Reagent for PYR test” and wait another 30 sec. A positive result is indicated by the red colour of the reaction zone. This test is again positive in S. pyogenes (and in Enterococcus, as well). Negative result can be seen in S. agalactiae and in non-A-non-B streptococci. Note: Formerly bacitracin test was used instead of the PYR test. Its principle was identical with that of the optochin test, only with another type of antibiotic. Due to its low specificity, it’s not in use any more. Fill in the following table, including drawing a result of the PYR test in all the three tested strains. Strain (write the letter) Strain (write the letter) Strain (write the letter) Interpretation: negative – positive (delete as appropriate) Interpretation: negative – positive (delete as appropriate) Interpretation: negative – positive (delete as appropriate) b) CAMP test Note: This test has nothing to do with cyclic adenosinmonophosphate, therefore it is CAMP test and not cAMP test. Its name is derived from the names of its inventors. The CAMP test is based on haemolytical synergism between S. aureus beta-haemolysin producing strain, and S. agalactiae strain. The positive result has the form of two triangular zones (“butterfly shape”) of complete haemolysis at the crossing of both strains. A small zone of a different shape is considered negative. Draw your result (the picture is on the following page): c) Demonstration of agglutination test for the detailed diagnostics of mainly non-A-non-B streptococci Both CAMP test and bacitracin and/or PYR test negative strains belong to the “non-A-non-B” group. Observe the result of the streptococcal agglutination from your dataprojection. Now, write the results of tasks 5 a), b) and c) in the table, and after that, make a final conclusion of tasks 1–5. Task 6: Antibiotic susceptibility tests in streptococci Evaluate the susceptibility tests (diffusion disc tests) for antibiotics in the strains of streptococci that you consider to be pathogens or possible pathogens (for the sake of simplification, consider the strains as originating from the upper respiratory tract). For the strain determined as a “non-A-non-B” streptococcus we do not perform the test, as its pathogenicity is low; for the strain determined as S. agalactiae (usually UTI origin) we have to use a special set of antibiotic, containing also special drugs for UTI treatment (e. g. nitrofurantoin). In the table, write the abbreviations of the antibiotics and for all the tested strains, measure the susceptibility zone in mm. Compare values measured by yourselves with the borderline values of diameters – according to these, interpret the zones as susceptible (S), resistant (R) or (if your zone equals to reference zone) dubious (D). Strain (letter) Strain (letter) Antibiotic Zone Æ (mm) Interpr. Zone Æ (mm) Interpr. Antibiotic Zone Æ (mm) Interpr. Penicillin (P) (ref. zone 18 mm) Penicillin (P) (ref. zone 18 mm) Erythromycin (E) (ref. zone 21 mm) Tetracycline* (TE) (ref. zone 23 mm) Clindamycin (DA) (ref. zone 17 mm) Vancomycin (VA) (ref. zone 13 mm) Chloramphenicol (C) (ref. zone 21 mm) Nitrofurantoin (F) (ref. zone 15 mm) Tetracycline* (TE) (ref. zone 23 mm) Vancomycin (VA) (ref. zone 13 mm) *result is also valid for doxycycline Task 7: Diagnostics of late sequels of streptococcal infections – ASO determination Principle – repetition of J08: Antibodies prevents hemolysin (streptolysin O – i.e. antigen) to hemolyse rabbit RBC. ASO levels increase after hemolytic streptococci group A (less commonly also other groups) caused infections. In risk for late sequellae, ASO increase over 200 I. U. (international units) is seen. On a side table, you will find a microtitration plate in a wet chamber. It includes a positive control and several sera. Determine the ASO values (ASO value = the last positive well; absence of haemolysis means positivity, haemolysis means negativity) and interprete the risk of late sequellae of streptococcal infections. Vocabulary to this topic: In this protocol (and some textbooks) In some other textbooks viridation alpha-haemolysis partial haemolysis beta-haemolysis total haemolysis no haemolysis/absence of haemolysis gamma-haemolysis